Achromatopsia is an autosomal recessive retinal disease characterized by markedly reduced visual acuity, nystagmus, severe photophobia under daylight conditions, and reduced or complete loss of color discrimination (Kohl, S. et al. Achromatopsia. In: Pagon R A, Bird T C, Dolan C R, Stephens K, editors Gene Reviews [Internet]. Seattle: University of Washington; 2010). It may be partial or complete. See Pang, J.-J. et al. (2010). Achromatopsia as a Potential Candidate for Gene Therapy. In Advances in Experimental Medicine and Biology, Volume 664, Part 6, 639-646 (2010) (hereinafter Pang et al). Symptoms of achromatopsia include reduced visual acuity, achromatopia (lack of color perception), hemeralopia (reduced visual capacity in bright light accompanied by photoaversion, meaning a dislike or avoidance of bright light), nystagmus (uncontrolled oscillatory movement of the eyes), iris operating abnormalities, and impaired stereovision (inability to perceive three-dimensional aspects of a scene). Electroretinograms reveal that in achromatopsia, the function of retinal rod photoreceptors remains intact, whereas retinal cone photoreceptors are not functional. Mutations in the cone-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene account for about 50% of cases of achromatopsia (Kohl S, et al. Eur J Hum Genet. 2005; 13:302-8). The rod and cone photoreceptors serve functionally different roles in vision. Pang et al. (2010). Cone photoreceptors are primarily responsible for central, fine resolution and color vision while operating in low to very bright light. They are concentrated in the central macula of the retina and comprise nearly 100% of the fovea. Rod photoreceptors are responsible for peripheral, low light, and night vision; they are found primarily outside the macula in the peripheral retina.
Approximately 1 in 30,000 individuals suffers from complete achromatopsia. In complete achromatopsia, there is total color vision loss, central vision loss, and visual acuity of 20/200 or worse. Thus, most individuals with achromatopsia are legally blind. The current standard of care consists of limiting retinal light exposure with tinted contact lenses and providing magnification to boost central vision. However, there is no treatment available that corrects cone function in achromatopsia. Pang et al.
There are various genetic causes of congenital achromatopsia. Mutations in the cyclic nucleotide-gated ion channel beta 3 (CNGB3, also known as ACHM3) gene, are one genetic cause of achromatopsia. Recent studies in dogs suggest some promise for the use of recombinant adeno-associated virus (rAAV)-based gene therapy for the treatment of achromatopsia caused by mutations in the CNGB3 gene. Komaromy et al., Gene therapy rescues cone function in congenital achromatopsia. Human Molecular Genetics, 19(13): 2581-2593 (2010) (hereinafter Komaromy et al.). In the canine studies, the rAAV vectors that were used packaged a human CNGB3 (hCNGB3) expression cassette that contained elements including a 2.1 kb cone red opsin promoter (PR2.1) and a human CNGB3 (hCNGB3) cDNA. One limitation of the studies is that the hCNGB3 driven by the PR2.1 promoter is expressed only in red and green cones, whereas endogenous hCNGB3 is expressed in all three types of cones (red, green and blue cones). Another limitation is that the overall size of the expression cassette utilized (5,230 bp) was well beyond the normal packaging capacity (<4.9 kb) of AAV particles; the over-stuffed rAAV particles dramatically impaired the rAAV packaging efficiency, resulting in low yields, a higher empty-to-full particle ratio, and likely a lower infectivity of the vector. Expression cassettes containing a shorter version of the cone red opsin promoter, or a cone arrestin promoter, were much less effective in restoring visual function. The present invention addresses these limitations.
The present invention has the advantage of providing promoters that are capable of promoting hCNGB3 expression in all three types of cones. In addition, the promoters of the invention have the advantage that they are short enough to make the hCNGB3 expression cassette fit well within the normal packaging capacity of rAAV. A promoter that fits within the normal rAAV packaging capacity provides benefits, such as improved yields, a lower empty-to-full particle ratio, and higher infectivity of the vector. The present invention also provides expression cassettes, vectors and kits that utilize these improved promoters, as well as methods for treating achromatopsia by administering the vectors.
The present invention addresses the need for an effective achromatopsia treatment.